Trichosanthin (TCS) is the active gradient of Chinese traditional medicine TIANHUAFEN, which is isolated from the root tuber of liana Trichosanthes kirilowii Maxim. TCS possesses multiple biological activities including abortifacient, immunoregulation, anti-tumor, and antiviral functions. In clinic, TCS exhibits significant effects on the treatment of abnormal proliferation of cytotrophoblast cells, such as hydatidiform mole, malignant hydatidiform mole and choriocarcinoma. In 1989, the discovery of the anti-HIV activity of TCS drew attentions worldwide.
TCS (GeneBank Accession number AY669811, SEQ ID NO: 8) comprises 247 amino acids with the molecular weight of about 27 kD and the pI of 9.4. It is a simple protein, without any glycosylation or other modifications. TCS belongs to type-I ribosome-inactivating proteins (RIPs) that can inhibit protein translation by specifically hydrolyzing the N—C glycosidic bond at 4324-adenosine of ribosomal 28S RNA of eukaryote. TCS can break supercoiled DNA and induce tumor cell apoptosis in vitro via caspase pathway. The main reason for TCS to kill tumor cells is the ribosome-inactivating and apoptosis-inducing activities.
It is believed that only after entering into target cells, can TCS play cytotoxic function, but the pathway by which type-I RIPs entering into cells is not clear. Recently, it was reported that the LDL-receptor family members played important roles in the cell entry of TCS, but the critical domain in TCS molecule interacting with its receptor is still undiscovered.
The future of TCS in clinical application is promising because of its medicinal value. However, as a protein derived from plant, the immunogenicity and side-effects of TCS are serious. Its clinical responses, including fever, urticaria, edema, muscle pains, and even effects on nerve centre, limit the application of TCS.
The side-effects of TCS come from two aspects. One is the immunogenecity of the epitopes on the surface of TCS molecule, and the other is the cytotoxicity on normal cells after TCS enters tissue cells. To decrease the side-effects of TCS, two considerations are proven to be effective: a) to remove the epitopes of TCS; and b) to change the cell entry of TCS by modifying the critical sites for TCS cell entry. Because TCS has several widely distributed epitopes, it is difficult to remove all of them. In contrast, the modification on the key sites of TCS cell entry can make TCS enter target tumor cells specifically without entering into normal tissue cells, so as to decrease the side-effects of TCS and immunogenecity by affecting antigen presentation of antigen presenting cells.
Currently, most researches on TCS are focusing on the relationship between TCS structure and activities. Researches on the side-effects and immunogenecity of TCS, especially on the cell identification and TCS entry, however, are rarely reported.
In 2002, Wang J H et al. reported that two TCS mutants, Lys120-Ile121-Arg122-Glu123 (SEQ ID NO: 9) to Ser120-Ala121-Gly122-Gly123 (SEQ ID NO: 10) and TCSE160A/E189A, showed much lowered ribosome inactivating (RI) activity and loss of anti-HIV activity. The mutant of R122G showed anti-HIV activity but its RI activity decreased 160 times. In 2003, Zhang F et al. reported that the deletion of 7 residues on the C-terminus of TCS decreased the cytotoxicity in vivo and RI activity in vitro, with RI activity decreased to a greater extent.
At present, targeting drugs show great potential in the clinical treatment of carcinoma and other diseases. The targeting drugs are composed of a targeting moiety that is generally a monoclonal antibody or antibody fragment (e.g. ScFv) specifically against tumor cells or virus-infected cells, and a toxin moiety that has anti-tumor or antiviral activity. The targeting moiety and toxin are ligated by chemical couplers or gene-fusion to produce targeting drugs with specific anti-tumor or anti-virus roles.